Recombinational cloning using engineered recombination sites

• US 6,720,140 B1
• Filed: 02/04/2000
• Issued: 04/13/2004
• Est. Priority Date: 06/07/1995
• Status: Expired due to Fees

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Invitrogen Corp. (Thermo Fisher Scientific Incorporated)
• Inventors: Brasch, Michael A., Hartley, James L.
• Primary Examiner(s): McKelvey, Terry
• Assistant Examiner(s): VOGEL, NANCY TREPTOW
• Application Number:
  US09/498,074
• Time in Patent Office: 1,530 Days
• Field of Search: 435/6, 435/320.1, 435/462, 536/23.1, 536/24.1
• US Class Current: 435/6.18
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12N 15/64 General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
• CPC Class Codes: C12N 15/66 General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
• CPC Class Codes: C12N 9/00 Enzymes; Proenzymes; Compositions thereof preparations containing enzymes for cleaning teeth A61K8/66, A61Q11/00; medicinal preparations containing enzymes or proenzymes A61K38/43; enzyme containing detergent compositions C11D; enzymes with nucleic acid structure, e.g. ribozymes, C12N15/113; Processes for preparing, activating, inhibiting, separating or purifying enzymes preparation of malt C12C1/00

Abstract

Recombinational cloning is provided by the use of nucleic acids, vectors and methods, in vitro and in vivo, for moving or exchanging segments of DNA molecules using engineered recombination sites and recombination proteins to provide chimeric DNA molecules that have the desired characteristic(s) and/or DNA segment(s).

Claims

1. A composition comprising one or more isolated recombination proteins and at least one isolated nucleic acid molecule comprising at least a first recombination site containing at least one mutation, wherein said mutation removes one or more stop codons from said recombination site.

3. The composition of claim 1 or claim 2, wherein said composition comprises at least two recombination proteins.

4. The composition of claim 1 or claim 2, wherein said composition comprises at least three recombination proteins.

2. A composition comprising one or more isolated recombination proteins and at least one isolated nucleic acid molecule comprising at least a first recombination site containing at least one mutation, wherein said mutation avoids hairpin formation.

5. A composition comprising at least two isolated recombination proteins, at least one Insert Donor DNA molecule comprising a first recombination site and a second recombination site wherein said first and second recombination sites do not recombine with each other, and at least one Vector Donor DNA molecule comprising a first recombination site and a second recombination site wherein said first and second recombination sites do not recombine with each other, wherein said Vector Donor DNA molecule further comprises (a) a repression cassette encoding a repressor and (b) a Selectable marker that is repressed by the repressor, and wherein the Selectable marker and the repression cassette are on different DNA segments, the DNA segments being separated from each other by at least one recombination site.

6. A composition comprising one or more recombination proteins and at least one isolated nucleic acid molecule, said nucleic acid molecule comprising at least a first att recombination site which comprises a core region having at least one mutation that enhances recombination efficiency or specificity in vitro in the formation of a Cointegrate DNA or a Product DNA molecule.

7. The composition of claim 6, wherein said recombination site confers at least one enhancement selected from the group consisting of (i) enhancing excisive recombination;

(ii) enhancing integrative recombination;

(iii) decreasing the requirement for host factors;

(iv) increasing the efficiency of the formation by recombination of said Cointegrate DNA or of said Product DNA;

(v) increasing the specificity of the formation by recombination of said Cointegrate DNA or of said Product DNA; and

(vi) increasing the specificity or yield of a subsequent recombination reaction of, or subsequent isolation of, the Product DNA.

8. The composition of claim 6, wherein said core region comprises a nucleic acid sequence selected from the group consisting of:

a) RKYCWGCTTTYKTRTACNAASTSGB (m-att) (SEQ ID NO;

1);

b) AGCCWGCTTTYKTRTACNAACTSGB (m-attB) (SEQ ID NO;

2);

c) GTTCAGCTTTCKTRTACNAACTSGB (m-attR) (SEQ ID NO;

3);

d) AGCCWGCTTTCKTRTACNAAGTSGB (m-attL) (SEQ ID NO;

4);

e) GTTCAGCTTTYKTRTACNAAGTSGB (m-attP1) (SEQ ID NO;

5);

and a corresponding or complementary DNA or RNA sequence, wherein R=A or G;

K=G or T/U;

Y=C or T/U;

W=A or T/U;

N=A or C or G or T/U;

S=C or G; and

B=C or G or T/U.

9. The composition of claim 6, wherein said core region comprises a nucleic acid sequence selected from the group consisting of:

a) AGCCTGCTTTTTTGTACAAACTTGT (attB1) (SEQ ID NO;

6);

b) AGCCTGCTTTCTTGTACAAACTTGT (attB2) (SEQ ID NO;

7);

c) ACCCAGCTTTCTTGTACAAACTTGT (attB3) (SEQ ID NO;

8);

e) GTTCAGCTTTCTTGTACAAACTTGT (attR2) (SEQ ID NO;

10);

f) GTTCAGCTTTCTTGTACAAAGTTGG (attR3) (SEQ ID NO;

11);

g) AGCCTGCTTTTTTGTACAAAGTTGG (attL1) (SEQ ID NO;

12);

h) AGCCTGCTTTCTTGTACAAAGTTGG (attL2) (SEQ ID NO;

13);

i) ACCCAGCTTTCTTGTACAAAGTTGG (attL3) (SEQ ID NO;

14);

j) GTTCAGCTTTTTTGTACAAAGTTGG (attP1) (SEQ ID NO;

15);

k) GTTCAGCTTTCTTGTACAAAGTTGG (attP2, P3) (SEQ ID NO;

16);

and a corresponding or complementary DNA or RNA sequence.

10. A kit for in vitro cloning of DNA segments comprising one or more isolated recombination proteins and at least one isolated nucleic acid molecule comprising at least a first recombination site containing at least one mutation, wherein said mutation removes one or more stop codons from said recombination site.

12. The kit of claim 10 or claim 11, wherein said kit comprises at least two recombination proteins.

13. The kit of claim 10 or claim 11, wherein said kit comprises at least three recombination proteins.

11. A kit for in vitro cloning of DNA segments comprising one or more isolated recombination proteins and at least one isolated nucleic acid molecule comprising at least a first recombination site containing at least one mutation, wherein said mutation avoids hairpin formation.

14. A kit comprising at least two isolated recombination proteins, at least one Insert Donor DNA molecule comprising a first recombination site and a second recombination site wherein said first and second recombination sites do not recombine with each other, and at least one Vector Donor DNA molecule comprising a first recombination site and a second recombination site wherein said first and second recombination sites do not recombine with each other, wherein said Vector Donor DNA molecule further comprises (a) a repression cassette encoding a repressor and (b) a Selectable marker that is repressed by the repressor, and wherein the Selectable marker and the repression cassette are on different DNA segments, the DNA segments being separated from each other by at least one recombination site.

15. A method for in vitro cloning comprising:

(a) mixing in vitro a first vector comprising at least a first recombination site and a second vector comprising at least a second recombination site; and

(b) incubating said mixture in the presence of at least one recombination protein under conditions sufficient to cause recombination of at least said first and second recombination sites,

wherein said first and/or second recombination site comprises at least a first nucleic acid sequence selected from the group consisting of a nucleic acid sequence that is 80-99% homologous to one or more of SEQ ID NOs;

1-16, a complementary DNA sequence thereto, and an RNA sequence corresponding thereto.

16. A method for in vitro cloning comprising:

(a) mixing in vitro a first vector comprising at least a first recombination site and a second vector comprising at least a second recombination site; and

(b) incubating said mixture in the presence of at least one recombination protein under conditions sufficient to cause recombination of at least said first and second recombination sites,

wherein said first and/or second recombination site comprises at least a first nucleic acid sequence selected from the group consisting of a nucleic acid sequence that hybridizes under stringent conditions to one or more of SEQ ID NOs;

1-16, a complementary DNA sequence thereto, and an RNA sequence corresponding thereto.

17. A method for in vitro cloning comprising:

(a) mixing in vitro a first vector comprising at least a first recombination site and a second vector comprising at least a second recombination site; and

(b) incubating said mixture in the presence of at least one recombination protein under conditions sufficient to cause recombination of at least said first and second recombination sites,

wherein said first and/or second recombination site comprises at least a first nucleic acid sequence selected from the group consisting of a mutated att recombination site containing at least one mutation that enhances recombinational specificity, a complementary DNA sequence thereto, and an RNA sequence corresponding thereto.

18. The method of claim 17, wherein said mutated att site comprises at least one nucleic acid sequence that is 80-99% homologous to at least one nucleic acid sequence as set forth in SEQ ID NOs.:

1-16.

19. The method of claim 17, wherein said mutated att site comprises at least one nucleic acid sequence that hybridizes under stringent conditions to at least one nucleic acid sequence as set forth in SEQ ID NOs.:

1-16.

20. A method of making a reaction mixture, comprising mixing at least one isolated recombination protein and at least one isolated nucleic acid molecule comprising at least a first recombination site containing at least one mutation, wherein said mutation removes one or more stop codons from said recombination site.

21. A reaction mixture made by the method of claim 20.

22. A method of making a reaction mixture, comprising mixing at least one isolated recombination protein and at least one isolated nucleic acid molecule comprising at least a first recombination site containing at least one mutation, wherein said mutation avoids hairpin formation.

23. A reaction mixture made by the method of claim 22.

24. A method of making a reaction mixture, comprising mixing at least one isolated recombination protein and at least one isolated nucleic acid molecule comprising at least a first recombination site containing a nucleic acid sequence that is 80-99% homologous to one or more of SEQ ID NOs:

1-16, a complementary DNA sequence thereto, and an RNA sequence corresponding thereto.

25. A reaction mixture made by the method of claim 24.

26. A method of making a reaction mixture, comprising mixing at least one isolated recombination protein and at least one isolated nucleic acid molecule comprising at least a first recombination site containing a nucleic acid sequence that hybridizes under stringent conditions to one or more of SEQ ID NOs:

1-16, a complementary DNA sequence thereto, and an RNA sequence corresponding thereto.

27. A reaction mixture made by the method of claim 26.

28. A method of making a reaction mixture, comprising mixing at least one isolated recombination protein and at least one isolated nucleic acid molecule comprising at least a first mutated att recombination site containing at least one mutation that enhances recombination specificity.

29. The method of claim 28, wherein said mutated att site comprises at least one nucleic acid sequence as set forth in SEQ ID NOs.:

1-16.

30. The method of claim 28, wherein said mutated att site comprises at least one nucleic acid sequence that is 80-99% homologous to at least one nucleic acid sequence as set forth in SEQ ID NOs.:

1-16.

31. The method of claim 28, wherein said mutated att site comprises at least one nucleic acid sequence that hybridizes under stringent conditions to at least one nucleic acid sequence as set forth in SEQ ID NOs.:

1-16.

32. A reaction mixture made by the method of claim 28.

33. A composition comprising at least one isolated recombination protein and at least one isolated nucleic acid molecule comprising at least a first recombination site containing at least one nucleic acid sequence selected from the group consisting of a nucleic acid sequence that is 80-99% homologous to one or more of SEQ ID NOs:

1-16, a complementary DNA sequence thereto, and an RNA sequence corresponding thereto.

34. A composition comprising at least one isolated recombination protein and at least one isolated nucleic acid molecule comprising at least a first recombination site containing at least one nucleic acid sequence selected from the group consisting of a nucleic acid sequence that hybridizes under stringent conditions to one or more of SEQ ID NOs:

1-16, a complementary DNA sequence thereto, and an RNA sequence corresponding thereto.

35. A composition comprising at least one isolated recombination protein and at least one isolated nucleic acid molecule comprising at least a first recombination site containing at least one nucleic acid sequence selected from the group consisting of a mutated attrecombination site containing at least one mutation that enhances recombinational specificity, a complementary DNA sequence thereto, and an RNA sequence corresponding thereto.

36. The composition of claim 35, wherein said mutated att site comprises at least one nucleic acid sequence as set forth in SEQ ID NOs.:

1-16.

37. The composition of claim 36, wherein said mutated att site comprises at least one nucleic acid sequence that is 80-99% homologous to at least one nucleic acid sequence as set forth in SEQ ID NOs.:

1-16.

38. The composition of claim 36, wherein said mutated att site comprises at least one nucleic acid sequence that hybridizes under stringent conditions to at least one nucleic acid sequence as set forth in SEQ ID NOs.:

1-16.

39. A kit for in vitro cloning of DNA segments comprising at least one isolated recombination protein and at least one isolated nucleic acid molecule comprising at least a first recombination site containing at least one nucleic acid sequence selected from the group consisting of a nucleic acid sequence that is 80-99% homologous to one or more of SEQ ID NOs:

1-16, a complementary DNA sequence thereto, and an RNA sequence corresponding thereto.

40. A kit for in vitro cloning of DNA segments comprising at least one isolated recombination protein and at least one isolated nucleic acid molecule comprising at least a first recombination site containing at least one nucleic acid sequence selected from the group consisting of a nucleic acid sequence that hybridizes under stringent conditions to one or more of SEQ ID NOs:

1-16, a complementary DNA sequence thereto, and an RNA sequence corresponding thereto.

41. A kit for in vitro cloning of DNA segments comprising at least one isolated recombination protein and at least one isolated nucleic acid molecule comprising at least a first recombination site containing at least one nucleic acid sequence selected from the group consisting of a mutated att recombination site containing at least one mutation that enhances recombinational specificity, a complementary DNA sequence thereto, and an RNA sequence corresponding thereto.

42. The kit of claim 41, wherein said mutated att site comprises at least one nucleic acid sequence as set forth in SEQ ID NOs.:

1-16.

43. The kit of claim 41, wherein said mutated att site comprises at least one nucleic acid sequence that is 80-99% homologous to at least one nucleic acid sequence as set forth in SEQ ID NOs.:

1-16.

44. The kit of claim 41, wherein said mutated att site comprises at least one nucleic acid sequence that hybridizes under stringent conditions to at least one nucleic acid sequence as set forth in SEQ ID NOs.:

1-16.

45. A method for in vitro cloning of a nucleic acid molecule, comprising:

(a) mixing in vitro a first vector comprising at least a first and second recombination sites, and a second vector comprising at least a third and fourth recombination sites, wherein said first and/or second vector further comprises a nucleic acid molecule to be cloned, and wherein said first and second recombination sites do not recombine with each other and said third and fourth recombination sites do not recombine with each other;

(b) incubating said mixture in the presence of at least one recombination protein under conditions sufficient to cause recombination of at least said first and third and/or said second and fourth recombination sites, thereby producing a product molecule comprising said nucleic acid molecule;

(c) contacting one or more hosts with said mixture; and

(d) selecting for a host comprising said product molecule, and against a host comprising said first vector and against a host comprising said second vector.

46. The method of claim 45, wherein said at least one recombination protein is selected from the group consisting of Int, IHF, Xis and Cre.

47. The method of claim 45, wherein said at least one recombination protein is selected from the group consisting of Int, IHF and Xis.

48. The method of claim 45, wherein said at least one recombination protein is selected from the group consisting of γ

δ

, Tn3, Tn7, resolvase, Hin, Gin, Cin and Flp.

49. The method of claim 45, wherein said at least one recombination protein is Cre.

50. The method of claim 45, wherein said first, second, third and/or fourth recombination sites are selected from the group consisting of att and lox P sites.

51. The method of claim 45, wherein said first, second, third and/or fourth recombination sites are att sites.

52. The method of claim 45, wherein said first, second, third and/or fourth recombination sites are lox P sites.

53. The method of claim 45, wherein said host is E. coli.

54. The method of claim 45, wherein said selection is accomplished using at least one Selectable marker.

55. The method of claim 54, wherein said Selectable marker is selected from the group consisting of an antibiotic resistance gene and a toxic gene.